The ubiquitin conjugation system (UCS) is a major pathway for the degradation of cellular proteins in eukaroytic cells and in some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. The UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes, such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994) Cell 79:13-21).
The process of ubiquitin conjugation and protein degradation occurs in four principal steps (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First ubiquitin (Ub), a small, heat stable protein (76 amino acids) is activated by a ubiquitin-activating enzyme (E1) in an ATP dependent reaction which binds the C-terminus of Ub to the thiol group of an internal cysteine residue in E1. Second, activated Ub is transferred to one of several Ub-conjugating enzymes (E2). Different ubiquitin-dependent proteolytic pathways employ structurally similar, but distinct ubiquitin-conjugating enzymes that are associated with recognition subunits which direct them to proteins carrying a particular degradation signal. E2 then links the Ub molecule through its C-terminal glycine to an internal lysine (acceptor lysine) of a target protein. In some instances accessory factors, known as ubiquitin-ligases or E3's, are required to work in conjunction with E2's for recognition of certain substrates. Additional Ub molecules may be added forming a multi-Ub chain structure. The ubiquinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and Ub is released for reutilization.
Prior to activation, Ub is usually expressed as a fusion protein composed of an N-terminal ubiquitin and a C-terminal extension protein (CEP), or as a polyubiquitin protein with Ub monomers attached head to tail. CEPs bear similarities to a variety of nucleic acid binding regulatory proteins. Most are highly basic with up to 30% lysine and arginine residues, and many have nucleic acid-binding, zinc-finger domains (Monia, B. P. et al. (1989) J. Biol. Chem. 264:4093-4103). These Ub-CEP fusion proteins are rapidly processed by C-terminal hydrolases which cleave Ub from the C-terminal side releasing free Ub for the UCS. The fusion of Ub with CEPs may allow co-regulation of the UCS with the translational activity of the cell. Processing of the CEPs may also be required to localize CEPs or Ub to specific cellular sites where they carry out their function (Monia et al., supra).
The E2 (Ub-conjugating) enzymes are important for substrate specificity in different UCS pathways. All E2s have a conserved domain of approximately 16 kDa called the UBC domain that is at least 35% identical in all E2s and contains a centrally located cysteine residue required for ubiquitin-enzyme thiolester formation (Jentsch, supra). A highly conserved proline-rich element is located N-terminal to the active cysteine residue. Structural variations beyond this conserved domain are used to classify the E2 enzymes. Class I E2s consist almost exclusively of the conserved UBC domain. This class includes the yeast E2's UBC4, 5, and 7. Class I E2's show only a weak ability to transfer ubiquitin to certain test proteins in vitro and may therefore require accessory E3 proteins for their activity in vivo (Jentsch, supra). UBC7 has also been shown to recognize ubiquitin itself as a substrate, thus forming polyubiquitin chains in vitro (van Nocker, S. et al. (1996) J. Biol. Chem. 271:12150-58). Class II E2s have various unrelated C-terminal extensions that contribute to substrate specificity and cellular localization. Yeast class II enzymes UBC2 and UBC3 have highly acidic C-terninal extensions that promote interactions with basic substrates such as histones. Yeast UBC6 has a hydrophobic signal-anchor sequence that localizes the protein to the endoplasmic reticulum.
Defects or alterations in the normal activity of the UCS are associated with a number of diseases and disorders. A decrease in muscle mass, known as muscle wasting or cachexia, has been shown to be associated with the UCS. Muscle wasting in tumor-bearing rats was associated with an increased ubiquitin-dependent proteolysis (Llovera M. et al. (1995) Int. J. Cancer 61: 138-141). The human tumor-suppressor protein p53 is degraded by the UCS and this process is mediated by an E2 enzyme in conjunction with a viral oncoprotein, E6-AP (Ciechanover, supra). Increased ubiquitin-dependent proteolysis has been detected in patients affected by neurodegenerative diseases such as Alzheimer's disease. Three extracellular forms of amyloid beta-protein (APP) precursor were degraded by this proteolytic pathway, suggesting a potential regulatory role for the ubiquitin-dependent system in the in vivo APP metabolic pathway (Gregori L. et al. (1994) Biochem. Biophys. Res. Commun. 203: 1731-1738). Abnormalities in the processing of APP are a possible cause of Alzheimer's disease.
Ubiquitin conjugation is a key rate-limiting step in antigen presentation (Grant E. P. et al. (1995) J. Immunol. 155: 3750-3758). Studies in mouse and rabbit reticulocytes indicated that the rates of degradation of beta-galactosidase constructs correlated with the rates of class I antigen presentation in vivo. Thus the ubiquitin degradation pathway may have a critical role in the immune response in generating major histocompatibility complex (MHC) class I-presented peptides.
The discovery of a new ubiquitin-conjugating enzyme and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer and immune and neurodegenerative disorders.